GSM7890932: Multiplexed scRNA-seq on WT and Snai1 KO gastruloids at 72h; Mus musculus; RNA-Seq
Gastruloids were collected, pooled in a 1.7 ml Eppendorf tube and washed twice in 1 ml of PBS. The number of gastruloids used in each condition was chosen to ensure that more than 300 000 cells were obtained. They were then dissociated in 100 µl of Accutase (Stempro) for 5 min at 37°C. Full dissociation was completed using mechanical dissociation and verified to ensure the absence of doublet cells. Cells were then resuspended in 200 µl of PBS and counted on a Countess 3 automated cell counter (Invitrogen). All centrifugations were done at 400g for 5 minutes. Some samples were then analysed with the standard 10x genomics pipeline (Chemistry v3 and v3.1), while others also included a CellPlex procedure to multiplex samples (see Supplementary Table 6 for list of samples and the method used). For the sample treated with the normal 10x procedure, after counting, dissociated cells were diluted in PBS BSA 0.04% (Millipore, A1595) to 1x106 cells/ml. For samples treated with the CellPlex multiplexing approach, cells were pelleted and incubated for 5 minutes in 50 µl of cell multiplexing oligos (3' CellPlex Kit Set A, PN-1000261) at room temperature. They were then washed three times with 1ml of PBS 1% BSA and finally they were resuspended in 110 µl of PBS 1% BSA and counted. The multiplexed samples were then pooled in equal proportions for each targeted sample. Both CellPlex and standard 10x Genomics sample were then filtered using a 40 µm cell strainer (Flowmi, BAH136800040). The pool of cells was counted and subjected to single cell RNA-seq following manufacturer's recommendation similarly as non-CellPlex samples (protocol CG000388 Rev A). For standard 10x samples, we aimed for a recovery of c.a. 6000 cells per sample while for multiplexed single cell RNA-seq we aimed for a recovery of maximum 24 000 cells as more doublet can be resolved with this method. cDNA preparations were performed according to 10x Genomics recommendations, amplified for 10–12 cycles and verified on fragment analyser. Both cell multiplexing oligo, and gene expression libraries were sequenced on a HiSeq 4000 (ref 15065681 v03) or a Novaseq (Illumina protocol #1000000106351 v03) with the cbot2 chemistry.